Guidelines for application of high-content screening in traditional Chinese medicine:concept,equipment,and troubleshooting

High-content screening(HCS)technology combines automated high-speed imaging hardware and single-cell quantitative analysis.It can greatly accelerate data acquisition in cellular fluorescence imaging and is a powerful research technique in traditional Chinese medicine(TCM).An increasing number of laboratories and platforms,including TCM laboratories,have begun utilizing HCS systems.However,this technology is still in its infancy in TCM research and there is a lack of sufficient experience with the associated concepts,instrument configurations,and analysis methods.To improve the understanding of HCS among researchers in the field of TCM,this paper summarizes the concept of HCS,software and hardware configuration,the overall research process,as well as common problems and related solutions of HCS in TCM research based on our team’s previous research experience,providing several research examples and an outlook on future perspectives,aiming to provide a technical guide for HCS in TCM research.

Screening potential mitochondria-targeting compounds from traditional Chinese medicines using a mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry method

Mitochondria regulate numerous crucial cell processes, including energy production, apoptotic cell death, oxidative stress, calcium homeostasis and lipid metabolism. Here, we applied an efficient mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry（LC/MS） method,also known as screening method for mitochondria-targeted bioactive constituents（SM-MBC）. This method allowed searching natural mitochondria-targeting compounds from traditional Chinese medicines（TCMs）, including Puerariae Radix（PR） and Chuanxiong Radix（CR）. A total of 23 active compounds were successfully discovered from the two TCMs extracts. Among these 23 hit compounds, 17 were identified by LC/MS, 12 of which were novel mitochondria-targeting compounds. Among these, 6 active compounds were analyzed in vitro for pharmacological tests and found able to affect mitochondrial functions. We also investigated the effects of the hit compounds on Hep G2 cell proliferation and on loss of cardiomyocyte viability induced by hypoxia/reoxygenation injury. The results obtained are useful for in-depth understanding of mechanisms underlying TCMs therapeutic effects at mitochondria level and for developing novel potential drugs using TCMs as lead compounds. Finally, we showed that SM-MBC was an efficient protocol for the rapid screening of mitochondria-targeting constituents from complex samples such as PR and CR extracts.

Ultrasonic Nebulization Extraction Coupled with Gas Chromatography-Mass Spectrometry for Analysis of Volatile Components in Traditional Chinese Patent Medicine

The ultrasonic nebulization extraction（UNE） was developed and applied to the extraction of volatile components from traditional Chinese patent medicine Xiaoyao Pills. Several parameters of ultrasonic nebulization extraction including the sample particle size, solvent volume, extraction time and ultrasonic power were studied and selected. As a result, 2.4 g of sample with particle size of 80 mesh was extracted with 15 mL of n-hexane for 20 min at an ultrasonic power of 35 W. The volatile components were analyzed by gas chromatography-mass spectrometry （GC-MS） under the optimal conditions and 57 compounds were identified. The precision, repeatability and stability of the proposed method were also studied. Compared with ultrasonic-assisted extraction（UAE） and hydrodistillation（HD） extraction, the proposed method is more efficient, faster and easier to be operated at room temperature with smaller sample and energy consumption. It is suggested that the ultrasonic nebulization extraction can be used as a novel alternative method for the extraction of volatile components from traditional Chinese patent medicine.

Screening of new non-nucleoside reverse transcriptase inhibitors of HIV-1 based on traditional Chinese medicines database

HIV- 1 RT is an important target for the treatment of AIDS. There are two major classes of antiviral agents that inhibit HIV- 1 RT have been identified, nucleoside RT inhibitors （NRTIs） and non-nucleoside RT inhibitors （NNRTIs）. In this report, a noval class of non-nucleoside compound with potential RT inhibitory activity were found from the traditional Chinese medicines database （TCMD） using a combination of virtual screening, docking, molecular dynamic simulations, where results were ranked by scoring function of the docking tool. The result indicates that M4753 （a compound derived from TCMD） has not only the lowest bonding energy but also the best match in geometric conformation with the forthcoming NNRTIs. Accordingly M4753 might possibly become a promising lead compound of NNRTIs for AIDS therapy.

Screening of Traditional Chinese Medicine Prescription for Treating Damp-heat Diarrhea of Piglets

[Objective] The paper was to explore the therapeutic effect of traditional Chinese medicine on damp-heat diarrhea of piglets in summer.[Method] Three prescriptions were developed with a dozen of traditional Chinese medicines,and in vitro antibacterial test against swine standard Escherichia coli [O149：K91,K88 ac（C83907）] was conducted.Moreover,106 natural cases of diarrhea piglets caused by coinfection of damp-heat and E.coli through clinical and laboratory diagnosis were then treated.[Result] The oral liquids prepared by three traditional Chinese medicine prescriptions had certain antibacterial effect on standard pathogenic E.coli.Prescription 2 presented the strongest inhibitory effects on swine E.coli in vitro,with the MIC of 31.25 mg/m L.It had better therapeutic effect,and the effective rate reached 94.74%.[Conclusion] The prescription 2 mainly consisted of Coptis chinensis and Prunus mume might be effective in treatment of piglet diarrhea caused by damp heat and E.coli in clinic.

Applications of HPLC/MS in the analysis of traditional Chinese medicines

In China, traditional Chinese medicines （TCMs） have been used in clinical applications for thousands of years. The successful hyphenation of high-performance liquid chromatography （HPLC） and mass spectrometry （MS） has been applied widely in TCMs and biological samples analysis. Undoubtedly, HPLC/MS technique has facilitated the understanding of the treatment mechanism of TCMs. We reviewed more than 350 published papers within the last 5 years on HPLC/MS in the analysis of TCMs. The present review focused on the applications of HPLC/MS in the component analysis, metabolites analysis, and pharmacokinetics of TCMs etc. 50% of the literature is related to the component analysis of TCMs, which show that this field is the most popular type of research. In the metabolites analysis, HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry has been demonstrated to be the powerful tool for the characterization of structural features and fragmentation behavior patterns. This paper presented a brief overview of the applications of HPLC/MS in the analysis of TCMs. HPLC/MS in the fingerprint analysis is reviewed elsewhere.

Application of Computer-Aided Drug Design to Traditional Chinese Medicine

Computer-aided drug design (CADD) is an interdisciplinary subject, playing a pivotal role during new drug research and development, especially the discovery and optimization of lead compounds. Traditional Chinese Medicine (TCM) modernization is the only way of TCM development and also an effective approach to the development of new drugs and the discovery of potential drug targets (PDTs). Discovery and validation of PTDs has become the “bottle-neck” restricted new drug research and development and is urgently solved. Innovative drug research is of great significance and bright prospects. This paper mainly discusses the “druggability” and specificity of PTDs, the “druglikeness” of drug candidates, the methods and technologies of the discovery and validation of PTDs and their application. It is very important to achieve the invention and innovation strategy “from gene to drug”. In virtue of modern high-new technology, especially CADD, combined with TCM theory, research and develop TCM and initiate an innovating way fitting our country progress. This paper mainly discusses CADD and their application to drug research, especially TCM modernization.

Drug discovery from traditional Chinese herbal medicine using high content imaging technology

Traditional Chinese medicine(TCM) has been widely used in China and other Asia countries for thousands of years to treat or prevent human diseases. Chinese herbal medicine, one of the most important components of TCM, has unique diversities in chemical components, and thus results in a wide range of biological activities. However, pharmaceutical industry is facing a major challenge to develop a large population of novel natural products and drugs, and considerable efforts have not resulted in highvolume of novel drug discovery and productivity. At present, increasing attention has been paid to Chinese herb medicine modernization in combination with the cutting-age technologies of drug discovery, especially the high throughput selection. High content imaging is an image-based high throughput screening method by using automated microscopy and image analysis software to capture and analyze phenotypes at a large scale to investigate multiple biological features simultaneously in the biological complex. Here, we described the pipeline of the state-of-the-art high content imaging technology, summarized the applications of the high content imaging technology in drug discovery from traditional Chinese herbal medicine, and finally discussed the current challenges and future perspectives for development of high throughput image-based screening technology in novel drug research and discovery.

Gaps and feasibility of fixed-dose combination strategy in the development of modern Chinese medicine

isTraditional Chinese medicine(TCM)is a highly complex chemical substance system,which not only reflected in the complexity of the chemical components and their interrelationships,but also in the intricacy of the prescription’s connection with the human body.Component compatibility strategy has been proposed for developing modern TCM since 2005 and established comprehensive relevant technologies and research approaches.Moreover,to meet the safety and efficacy of current pharmaceuticals,research on fixed-dose combination drugs is directed by modern scientific theories,conforms to TCM compatibility principles and clarifies material basis and pharmacological mechanisms and component-effect correlations.This review summarized gaps and feasibility of fixed-dose combination strategy in the development of modern TCM research and assessed their advantages and disadvantages in light of contemporary drug combination research practices.

Accurate construction of cell membrane biomimetic graphene nanodecoys via purposeful surface engineering to improve screening efficiency of active components of traditional Chinese medicine

Biomimetic nanoengineering presents great potential in biomedical research by integrating cell membrane(CM) with functional nanoparticles. However, preparation of CM biomimetic nanomaterials for custom applications that can avoid the aggregation of nanocarriers while maintaining the biological activity of CM remains a challenge. Herein, a high-performance CM biomimetic graphene nanodecoy was fabricated via purposeful surface engineering, where polyethylene glycol(PEG) was used to modifying magnetic graphene oxide(MGO) to improve its stability in physiological solution, so as to improve the screening efficiency to active components of traditional Chinese medicine(TCM). With this strategy, the constructed PEGylated MGO(PMGO) could keep stable at least 10 days, thus improving the CM coating efficiency. Meanwhile, by taking advantage of the inherent ability of He La cell membrane(HM) to interact with specific ligands, HM-camouflaged PMGO showed satisfied adsorption capacity(116.2 mg/g) and selectivity. Finally, three potential active components, byakangelicol, imperatorin,and isoimperatorin, were screened from Angelica dahurica, whose potential antiproliferative activity were further validated by pharmacological studies. These results demonstrated that the purposeful surfaceengineering is a promising strategy for the design of efficient CM biomimetic nanomaterials, which will promote the development of active components screening in TCM.

Characterization of multiple chemical components of GuiLingJi by UHPLC-MS and ^(1)H NMR analysis

GuiLingJi(GLJ),a classic traditional Chinese medicine(TCM)formula,is composed of over 20 herbs,according to the Pharmacopeia of the People's Republic of China.Owing to its various activities,GLJ has been used in clinical settings for more than 400 years in China.However,the ambiguous chemical material basis limits the development of studies on the quality control and pharmacological mechanisms of GLJ.Therefore,comprehensive characterization of the multiple chemical components of GLJ is of great significance for the modernization of this formula.Given the great variety of herbs in GLJ,both UHPLCMS and ^(1)H NMR techniques were employed in this study.In addition,solvent extraction with different polarities was used to eliminate signal interference and the concentration of trace components.A variety of MS analytic methods were also used,including implementation of a self-built compound database,diagnostic ion filtering,mass defect filtering,and Compound Discoverer 3.0 analysis software.Based on the above strategies,a total of 150 compounds were identified,including 5 amino acids,13 phenolic acids and glycosides,11 coumarins,72 flavones,20 triterpenoid and triterpenoid saponins,23 fatty acids,and 6 other compounds.Moreover,13 compounds were identified by ^(1)H NMR spectroscopy.The UHPLC-MS and ^(1)H NMR results supported and complemented each other.This strategy provides a rapid approach to analyzing and identifying the chemical composition of Chinese herbal prescriptions.The current study provides basis for further research on the quality control and pharmacological mechanism of GLJ.

Brain organoids are new tool for drug screening of neurological diseases

At the level of in vitro drug screening,the development of a phenotypic analysis system with highcontent screening at the core provides a strong platform to support high-throughput drug screening.There are few systematic reports on brain organoids,as a new three-dimensional in vitro model,in terms of model stability,key phenotypic fingerprint,and drug screening schemes,and particula rly rega rding the development of screening strategies for massive numbers of traditional Chinese medicine monomers.This paper reviews the development of brain organoids and the advantages of brain organoids over induced neurons or cells in simulated diseases.The paper also highlights the prospects from model stability,induction criteria of brain organoids,and the screening schemes of brain organoids based on the characteristics of brain organoids and the application and development of a high-content screening system.

Research Progress of Tea Functional Components in the Prevention and Treatment of Lung Cancer

The incidence and mortality of lung cancer rank first among all malignant tumors,so the prevention and treatment of lung cancer has become a worldwide concern.Tea is one of the most popular healthy drinks in the world.A large number of studies have shown that tea polyphenols,theophylline,tea pigments,theanine,tea polysaccharides and other functional components in tea can promote apoptosis of cancer cells,inhibit proliferation and division of cancer cells,and regulate the expression of tumor-related gene protein through strong antioxidant activity,inhibiting the activity of key enzymes and blocking signal transduction to prevent and treat the occurrence,development and metastasis of lung cancer.In this study,the application of tea in the prevention and treatment of lung cancer was elaborated through the traceability,basic components,basic theory of traditional Chinese medicine,anti-lung cancer mechanism,expert opinions and so on,in order to provide direction for the prevention and treatment of tea and lung cancer.

Tocolysis Effects of Traditional Chinese Medicine and Their Effective Components

Traditional Chinese medicine(TCM)for anti-abortion is based on the theory of gynecology of TCM,which aims to prevent and treat abdominal pain during pregnancy,fetal leakage,uneasy fetal movement,and fetal atrophy.Because of the complexity of the ingredients of Chinese Materia Medica,and the imprecise intervention mechanism for tocolysis,further investigation about the effects of Chinese herbs and their components on tocolysis by utilizing advanced technologies is required to be made.All the information available about TCM and its effects on pregnant women and fetuses was collected via electronic search using Web of Science,PubMed,and CNKI,and a library search was performed to locate classic herbal medicine books.The active ingredients in TCM were screened with the help of Traditional chinese medicine systems pharmacology database and analysis platform(TCMSP).The keywords being used included herbal names,pharmacology,pregnancy,threatened abortion,and fetus.Modern pharmacological studies have shown that TCM mainly prevents threatened abortion by a direct effect on the pregnant woman’s immune system,sex hormones such as estrogen and progesterone,the uterus and the endometrium,and the decidual tissue such as the placenta.It may also address pregnancy complications due to advanced maternal age,infection,polycystic ovary syndrome,diabetes,and mental disorders caused by threat of a miscarriage.TCM protects against spontaneous miscarriage,but its mechanisms are largely unknown.This research applies scientific methods to characterize and examine the effective components of TCM and their application to lower the risk of abortion to the pregnant women and fetuses.

CHEMISTRY AND BIOACTIVITY OF POLYSACCHARIDES FROM TRADITIONAL CHINESE MEDICINE MAHUANG

The long-term clinical practice of traditional Chinese medicine(TCM)confirms its importance and essential role in the health care system in China,especially in the prevention and treatment of chronic diseases.TCM is the holistic medicine under the guidance of system theory,emphasizing harmony between human and nature,focusing on equilibrium and balance,and focusing on

UPLC-QTOF-MS快速筛查与确证中药中马兜铃酸类物质

基于超高效液相色谱-四极杆飞行时间串联质谱技术,建立马兜铃酸类物质的高通量、高分辨筛查分析方法.对75个中草药、中成药等样品进行筛查,其中31种中草药、23种中成药、5种鱼腥草、9种鱼腥草滴眼液均有不同程度的马兜铃酸类物质检出.针对检出的6种马兜铃酸类物质建立定量方法,其线性相关系数R^(2)均大于0.994,各目标物的检测限在2.5~50μg·L^(-1)范围内,回收率在86.27%~106.89%范围内.该方法操作简便快速,适用于大批量样品的快速筛查分析.

Magnetic nanoparticles used in headspace extraction coupled with DSI-GC-IT/MS for analysis of VOCs in dry Traditional Chinese Medicine

A novel magnetic method using polystyrene modified magnetic nanoparticles to perform thermoheadspace extraction was successfully developed for extraction and preconcentration of volatile organic components in dry Traditional Chinese Medicine（TCM） based on gas chromatography-ion trap/mass spectrometry with a Chromato Probe direct sample introduction device. The dried fruit of Amomum testaceum Ridl. was used as the object TCM. The optimum parameters of headspace magnetic solid-phase extraction were investigated, in which desorption solvent ethyl acetate played a key role in this method,and the headspace extraction temperature of 90℃ and the headspace extraction time of 15 min finally decided. Headspace solid-phase microextraction method was also used to analyze volatile compounds in the TCM to compare with the proposed method. The results show that 60 components were identified totally by two methods; most of the low boiling point chemical compounds are isolated by this new method. In this work, an environmental-friendly and cheap analytical method was established, and a new approach to analyze volatile compounds in dry Traditional Chinese Medicine was also provided.

Bioactive constituents of animal-derived traditional Chinese medicinal materials for breast cancer:opportunities and challenges

Breast cancer is globally the most common invasive cancer in women and remains one of the leading causes of cancer-related deaths.Surgery,radiotherapy,chemotherapy,immunotherapy,and endocrine therapy are currently the main treatments for this cancer type.However,some breast cancer patients are prone to drug resistance related to chemotherapy or immunotherapy,resulting in limited treatment efficacy.Consequently,traditional Chinese medicinal materials(TCMMs)as natural products have become an attractive source of novel drugs.In this review,we summarized the current knowledge on the active components of animal-derived TCMMs,including Ophiocordyceps sinensis-derived cordycepin,the aqueous and ethanolic extracts of O.sinensis,norcantharidin(NCTD),Chansu,bee venom,deer antlers,Ostrea gigas,and scorpion venom,with reference to marked anti-breast cancer effects due to regulating cell cycle arrest,proliferation,apoptosis,metastasis,and drug resistance.In future studies,the underlying mechanisms for the antitumor effects of these components need to be further investigated by utilizing multi-omics technologies.Furthermore,large-scale clinical trials are necessary to validate the efficacy of bioactive constituents alone or in combination with chemotherapeutic drugs for breast cancer treatment.

Discrimination of Wild-Grown and Cultivated <i>Ganoderma lucidum</i>by Fourier Transform Infrared Spectroscopy and Chemometric Methods

Wild-grown Ganoderma lucidum (G. lucidum), a traditional Chinese herbal medicine, is highly cherished and expensive for its medicinal efficiency. This study targets the development of an accurate and effective analytical method to distinguish wild-grown G. lucidum from cultivated ones, which are of essential importance for the quality assurance and estimation of its medicinal value. Furthermore, different parts of G. lucidum have been studied to examine the differences between wild-grown and cultivated ones. Fourier transform infrared (FTIR) diffuse reflectance spectroscopy combined with the appropriate chemometric method has been proven to be a rapid and powerful tool for discrimination of wild-grown and cultivated G. lucidum with classification accuracy of 98%. The informative spectral absorption bands for discrimination emphasized by the linear diagnostic rule have provided quantitative interpretations of the chemical constituents of wild-grown G. lucidum regarding its anticancer effects.

Systematic review of robust experimental models of rheumatoid arthritis for basic research

Rheumatoid arthritis(RA)is a common autoimmune disease characterized by progressive joint inflammation and destruction,deformity,loss of mobility,and permanent disability.Although the cellular and molecular mechanisms involved in RA are understood in detail,no drugs or therapies can completely cure RA.Many long-term efforts have been directed towards a better understanding of RA pathogenesis and the development of new classes of therapeutics.Thus,the ongoing elucidation of pathogenic events underlying RA mostly relies on studies of animal models.Herein,we comprehensively review and discuss the characteristics,challenges,and unresolved of issues of various experimental models of RA to provide a basis and reference for the rational selection of experimental RA models for basic investigations into traditional Chinese medicine(TCM).